www.nature.com/scientificreports OPEN Selection of carbohydrate- active probiotics from the gut of carnivorous fsh fed plant-based Received: 4 February 2019 Accepted: 25 March 2019 diets Published: xx xx xxxx Cláudia R. Serra 1, Eduarda M. Almeida2,3, Inês Guerreiro1,2, Rafaela Santos 1,2, Daniel L. Merrifeld4, Fernando Tavares 2,3, Aires Oliva-Teles1,2 & Paula Enes1 The gastrointestinal microbiota plays a critical role on host health and metabolism. This is particularly important in teleost nutrition, because fsh do not possess some of the necessary enzymes to cope with the dietary challenges of aquaculture production. A main difculty within fsh nutrition is its dependence on fsh meal, an unsustainable commodity and a source of organic pollutants. The most obvious sustainable alternatives to fsh meal are plant feedstufs, but their nutritive value is limited by the presence of high levels of non-starch polysaccharides (NSP), which are not metabolized by fsh. The composition of fsh-gut microbial communities have been demonstrated to adapt when the host is fed diferent ingredients. Thus, we hypothesized that a selective pressure of plant-based diets on fsh gut microbiota, could be a benefcial strategy for an enrichment of bacteria with a secretome able to mobilize dietary NSP. By targeting bacterial sporulating isolates with diverse carbohydrase activities from the gut of European sea bass, we have obtained isolates with high probiotic potential. By inferring the adaptive ftness to the fsh gut and the amenability to industrial processing, we identifed the best two candidates to become industrially valuable probiotics. This potential was confrmed in vivo, since one of the select isolates lead to a better growth and feed utilization efciency in fsh fed probiotic- supplemented plant-based diets, thus contributing for sustainable and more cost-efective aquaculture practices. Te gastrointestinal microbial community is a critical actor which impacts host metabolism, immune status and health/disease balance. In the last decade, this relationship has received increased attention particularly in humans, where it is known to control local (at the gut level) health status as well as systemic health1–3. Te gut microbiota of vertebrates, ranging from mammals to teleost fsh, is involved in host appetite control and obesity development4, protection against pathogens, immunity enhancement or infammatory processes5–7. Additionally, gut microorganisms respond to a wide range of factors, including dietary composition, and harbor a relevant and diversifed enzymatic repertoire that might interfere with host metabolism8,9. Tis is particularly important in fsh nutrition, because fsh do not possess all of the necessary enzymes to cope with the current aquaculture dietary challenges10–15. Aquaculture output is growing rapidly that has already surpassed fsheries in terms of providing food to meet the growing human population16. Fish aquaculture is greatly dependent on fsh meal (FM), an unsustainable com- modity and a source of organic pollutants, almost exclusively provided by fsheries. Tis is particularly obvious in carnivorous fsh production due to their high dietary protein requirement (40–50%), which is mainly provided by FM. Plant feedstufs (PF) are sustainable alternatives to FM, and among them, soybean meal (SBM), rape- seed meal (RSM), and sunfower meal (SFM), have been acknowledged as the most promising due to their high 1CIMAR/CIIMAR - Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, s/n, 4450-208, Matosinhos, Portugal. 2Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, Ed. FC4, 4169- 007, Porto, Portugal. 3CIBIO - Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO, Laboratório Associado, Universidade do Porto, Campus Agrário de Vairão, 4485-661, Vairão, Portugal. 4School of Biomedical and Biological Sciences, Plymouth University, 401 Davy Building, Drake Circus, Plymouth, PL4 8AA, Devon, UK. Correspondence and requests for materials should be addressed to C.R.S. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:6384 | https://doi.org/10.1038/s41598-019-42716-7 1 www.nature.com/scientificreports/ www.nature.com/scientificreports protein level, world-wide availability, and reasonable price17,18. However, the nutritive value of PF is limited by the presence of several anti-nutritional factors, including high levels of non-starch polysaccharides (NSP) which are not digested by fsh17–20. NSP content in SBM, RSM, and SFM averages 22–24% and the major NSP compo- nents are pectic polysaccharides with arabinose, galactose, and xylose residues predominating21. Yet, the pro- portion of these sugar residues varies between PF with galactose being predominant in SBM, arabinose in RSM, and xylose in SFM21. In fsh, the carbohydrate-active enzymes (CAZymes) able to hydrolyze the β-glycosidic bonds of NSP are scarce or non-existent15. Tus, dietary NSP remain indigestible and cannot be used as energy source. Moreover, indigestible NSP might have detrimental efects on fsh performance and nutrient digestibility and on fsh health22. Tese adverse efects are associated with the viscous nature of NSP and their interaction with gut epithelium, mucus, and microbiota, which ultimately result on physiological and infammatory imbal- ances22. Additionally, and contrary to other animal species, such as pigs and poultry, the supplementation of PF based diets with exogenous carbohydrases does not necessarily translate into an efective strategy for improving NSP utilization, as diverging results on their impact on fsh growth performance and feed utilization have been reported23–26. Terefore, gut microorganisms characterized by a rich secretome are a potential source of in loco carbohydrases that may help fsh to overcome the mentioned constraints. Live microorganisms that confer a health beneft to the host when administered in adequate amounts are denominated probiotics27. Tese benefcial microbes might beneft the host, decreasing the incidence of dis- eases by competing with pathogens for adhesion sites/nutrients; producing natural antimicrobial compounds that inhibit pathogens growth; contributing to a balanced gut microbiota; improving host growth; enhancing host immune system and gastrointestinal histomorphology28. Probiotics have also been implicated in bioremediation and water quality improvement by reducing antibiotic usage, contributing to aquaculture sustainability28. Among the bacterial species currently used as probiotics, sporeformers show critical advantages: bacterial spores are remarkably resistant dormant structures29, permitting good shelf-storage; spores are easily produced in large scale and can be dehydrated, facilitating feed incorporation. Importantly, spores survive gut transit since they are acid and bile tolerant, and become successfully established in the gut30. In particular, Bacillus subtilis spores, which enjoy GRAS (Generally Regarded As Safe) status from the U.S. Food and Drug Administration (FDA) and are included in the European Food Safety Authority (EFSA) list of Qualifed Presumption of Safety (QPS)31, experience exponentially growing applications in biomedicine and biotechnology (as oral vaccines, dis- infectants, probiotics or display systems)32–36. In fact, diferent sporeformers are nowadays used as human and animal probiotics32,34,37,38, but within European Union just one probiotic has been authorised for use in aquacul- ture (Bactocell , LALLEMAND Inc., Canada). We hypothesize® that a selective pressure of diets on fsh gut microbiota is a hopeful strategy for an enrich- ment of bacteria with a secretome able to mobilize dietary components, including the capacity of NSP utiliza- tion. Several studies emphasize the presence of carbohydrate-active enzymes in sporeforming species such as Bacillus spp.39, but few reports exist on the isolation of carbohydrolytic sporeformers from fsh gut with probiotic potential that could be administrated in aquafeeds to help fsh in their digestive challenges11,40–42. Furthermore, available information on carbohydrases-producing bacteria is mainly restricted to amylolytic, cellulolytic and chitinolytic enzymes41–43. Tus, screening fsh gut microbiota for bacteria capable of producing extracellular digestive enzymes that hydrolyse NSP present in PF (such as mannans, glucans, xylans, arabinans, and galactans), is a promising and unexplored research topic. Gut microbiota isolates showing promising metabolic traits and absence of safety concerns can be used as probiotics in cost-efective and environmental-friendly diets by allow- ing the host to obtain energy from otherwise indigestible dietary constituents. In fact, native bacteria with pro- biotic potential will be more apt to become established and persist in the fsh gut environment afer withdrawal from the diet44. Tis study describes the isolation, identifcation and characterization of marine fsh gut sporeformers capable of producing carbohydrate-active enzymes that hydrolyse NSP and accesses their potential as probiotics for use in aquafeeds. Sporeformers were isolated from the gut of European sea bass (Dicentrarchus labrax) juveniles challenged with PF diets based on SBM, RSM or SFM, which have diferent NSP profles. European sea bass was the model species chosen due to its high commercial importance in European aquaculture and its carnivorous feeding habits, thus being